Intestinal cholesterol absorption plays a critical role in the maintenance of the body cholesterol homeostasis. This is inferred from studies showing a positive correlation between efficiency of intestinal cholesterol absorption and the level of plasma cholesterol. Additionally, pharmacological inhibition of intestinal cholesterol absorption by the recently discovered drug ezetimibe (Zetia) has been shown to be an effective therapy to lower plasma cholesterol. In this regard, the Niemann-Pick Type C1-Like 1 (NPC1L1) transporter represents the major pathway for intestinal cholesterol absorption. An increase in intestinal cholesterol absorption with concomitant upregulation of NPC1L1 expression has been implicated in the pathophysiology of hypercholesterolemia associated with diseases such as diabetes mellitus. Besides recent advances in therapeutic modalities to lower plasma cholesterol, treatment of hypercholesterolemia in patients with diabetes mellitus is still a challenge. Therefore, an understanding of the molecular mechanisms regulating NPC1L1 function and expression under physiological conditions and in diseased states would facilitate development of future therapeutic modalities for hypercholesterolemia. The major pathological feature of diabetes mellitus is insulin deficiency or a defective response to insulin. Whether insulin plays a role in NPC1L1 regulation under basal conditions or in diseased conditions of altered cholesterol homeostasis observed in diabetes mellitus is not known. We hypothesized that insulin decreases intestinal cholesterol absorption via the modulation of NPC1L1 function and expression and that the increase in intestinal cholesterol uptake and NPC1L1 expression in diabetes mellitus results from disturbances in the molecular pathways involved in NPC1L1 regulation. Our preliminary data utilizing human intestinal Caco2 monolayers demonstrated that NPC1L1 function, expression and promoter activity are decreased by insulin. We also demonstrated an increase in the expression of intestinal NPC1L1 in the rat model of Streptozotocin (STZ)-induced diabetes mellitus in parallel to an increase in cholesterol uptake in isolated enterocytes. However, the molecular mechanisms underlying regulation of NPC1L1 by insulin are not known. Therefore, our proposed studies represent a systematic approach designed to extensively investigate NPC1L1 regulation by insulin utilizing an in vitro cell culture model (Specific objective 1) and to examine the mechanisms of NPC1L1 alterations in diseases associated with hypercholesterolemia utilizing an in vivo rat model of diabetes mellitus (Specific Objective 2). Studies proposed in Specific objective 1 will utilize molecular biology techniques such as promoter analysis, DNAase footprinting and chromatin immunoprecipitation assays to delineate the transcriptional regulation of NPC1L1 by insulin. Involvement of signaling molecules and transcription factors in NPC1L1 regulation by insulin will be further elucidated by siRNA and other biochemical approaches. Specific objective 2 will focus on delineating the molecular regulation of NPC1L1 function and expression in rat model of diabetes mellitus utilizing the state-of-the art approaches such as cellular uptake in isolated enterocytes, esterification of cholesterol, laser capture microdissection and confocal microscopy. The results of the proposed studies will enhance our understanding of the regulation of the processes involved in intestinal cholesterol absorption by insulin and will reveal potential targets for better and more effective therapeutic modalities to manage hypercholesterolemia associated with diseases such as diabetes mellitus. Potential Impact on Veterans Health Care: The proposed studies are highly relevant to the VA mission since the prevalence of diabetes mellitus and cholesterol homeostasis disorders such as atherosclerosis are higher in the VA patient population than in the general population. The results of our studies will aid in the development of better therapeutic modalities for the treatment of hypercholesterolemia and hence improve the management of complications associated with diabetes mellitus. PUBLIC HEALTH RELEVANCE: An increase in intestinal absorption of cholesterol occurs in diseases such as diabetes mellitus leading to high plasma cholesterol and increased risk of cardiovascular diseases. Therefore, it is important to investigate the mechanisms underlying alterations in cholesterol absorption under physiological conditions and in diseases such as diabetes mellitus. The proposed studies focus on insulin mediated regulation of the cholesterol transporter, NPC1L1 and its modulation in rat model of diabetes mellitus. These studies have direct relevance to public and veterans health because of the high incidence of diabetes mellitus in both general and VA patient population and are of great importance for designing better therapeutic modalities for the management of cholesterol-related disorders in future.